Gram-negative bacterial septicemia is a common disorder with an unacceptably high morbidity and mortality. The syndrome is due, in part, to the interaction of the lipid A portion of bacterial lipopolysaccharide (LPS, endotoxin) with human leukocytes. Several lines of evidence point to the existence of specific lipid A recognition proteins (receptors) on the leukocyte membrane suggesting the possibility of receptor based therapy for endotoxin shock. The long term goal of this proposal is to define the pharmacology of lipid A receptors based on interrelated approaches: analysis of LPS responses with defined LPS agonists and antagonists; the generation of monoclonal antibodies which block lipid A receptors (antagonists); the use of ligand binding methodology to clone lipid A binding proteins; the generation and characterization of mutant cell lines deficient in binding or response to lipid A. This approach to defining lipid A receptors is based on the observation that in other receptor systems, pharmacologic studies with defined ligands, membrane biochemistry and the analysis of mutants have defined receptors and receptor subtypes which ultimately correlated with unique gene products. Short term responses in human neutrophils will be characterized in detail with newly available LPS antagonists. Novel monoclonal antibodies (mAbs) which antagonize the effects of LPS will be sought by immunizing mice with human monocytes and screening the resultant hybridoma supernatants for their ability to inhibit LPS induced release of tumor necrosis factor-alpha. To test the possibility that antigens identified by inhibitory mAbs might represent lipid A receptors, immunoaffinity purified proteins will be tested for lipid A binding. Antibodies with LPS antagonist activity might be therapeutic for patients with clinical sepsis. Using a related approach based on ligand binding, the PI seeks to use synthetic enzymatically labeled. [32P]-lipid A, unique in specific activity (~1010 dpm/nmole) and purity, to define and clone lipid A receptors. A cloning strategy is described whereby transiently COS cells expressing lipid A binding proteins are enriched on LPS coated plastic ("panning") and corresponding cDNA are selected by [32P]-lipid A binding. Two alternative approaches to cloning lipid A receptors are also outlined. Finally, an established mutant monocyte cell line deficient in the LPS binding protein CD18 will be characterized in order to learn the role CD18 plays in LPS induced signal transduction. New mutant cell lines either deficient in the expression of a defined surface protein or in a defined response to LPS will be sought. The characterization of such phenotypes may allow both an understanding of the complex pharmacology of LPS as well as a genetic analysis of LPS responsiveness. A detailed understanding of lipid A/animal cell membrane interactions should ultimately translate to an improved ability to design adjuvant therapies for gram-negative bacterial septicemia.